Abstract Hypoxic ischemic (HI) insult damages both white matter and grey matter in infants and causes significant mortality and morbidity. To investigate the pathological mechanisms of neonatal HI injury and find satisfactory treatments, animal models of neonatal hypoxic ischemic injury have been established and widely used. In this project, novel in vivo magnetic resonance imaging on tissue microstructure and neuronal activity will be developed to examine the progression of HI injury and the effects of therapeutic hypothermia in a neonatal mouse model. In aim 1, we will examine the sensitivity of novel diffusion MRI (dMRI) techniques to tissue microstructural changes caused by HI injury. Preliminary results have shown that the proposed imaging techniques can more sensitively detect mild brain injuries than conventional dMRI techniques and is less susceptible to confounding pseudo-normalization of conventional dMRI signals. We will use histology and electron microscopy to determine the levels of cellular and subcellular structural changes and correlate quantitative measurements with dMRI signals, and use numerical simulations to understand the relationships between them. The knowledge will be used to optimize the imaging protocols to detect key structural changes after neonatal HI. In aim 2, we will examine injury using manganese-enhanced MRI (MEMRI). Previous studies have shown that the MEMRI contrasts reflect neuronal activity in the brain and can selectively enhance regions with apoptosis and inflammation after neonatal HI. In this aim, we will examine the sensitivity of MEMRI to loss of neuronal activity, apoptosis, and inflammation after neonatal HI. With both dMRI and MEMRI, we will be able to examine a broad range of pathological events after neonatal HI. Hypothermia is the standard care for newborns with neonatal HI, but its protective mechanisms are not clearly understood. It has been assumed that hypothermia reduces cell swelling, inflammation, and vasogenic edema, and may delay the pseudo-normalization process. In aim 3, the techniques developed in the first two aims will then be applied to characterize HI injury in mice treated with hypothermia to quantitatively characterize its effects and elucidate its neuroprotective mechanisms. We expect the project to extend our knowledge on the relationships between pathology and diagnostic markers in this mouse model, and shed light on the mechanisms of HI injury and therapeutic hypothermia. This information and techniques developed in this project will be useful to design effective strategies for intervention and to monitor treatment response in studies using this or similar models.